Figure 3. TRAF1 is required for HIV-specific CD8 T cell responses. (a) CD8 and CD4 T cells were separately purified from HIV+ or HIV− donors. CD4 T cells were infected with a primary isolate of HIV and at 48 h, co-cultured with their autologous CD8 T cells that had been transfected with either TRAF1-specific siRNA or a control scrambled RNA (ctrl). Irradiated autologous PBMCs were added as a source of APC. The frequency of Gag+ T cells was measured 5–7 d later by flow cytometry using CD3-, CD8-, and GAG-specific antibodies to assess the proportion of infected CD4 T cells. As CD4 is down-regulated on the infected cells, the absence of CD8 is used to determine the CD4 T cell population. Representative flow cytometry plots are shown in Fig. S2. (left and middle) representative suppression curves (based on three to five replicates at each effector-to-target ratio for each donor and representative of three viral controllers and a healthy uninfected control). Statistical significance was determined by linear regression of percentage of Gag+ T cells against log (effector: target ratio) using GraphPad (Prism) software. (right) Representative Western blot analysis of TRAF1 levels after knockdown, determined at 48 h after activation. (b). (left and middle) Viral suppression assay performed as in a. Bim-specific siRNA, TRAF1-specific siRNA, or both, or control scrambled RNA were used to transfect CD8 T cells from two viral controllers. CD8 T cells were plated at a ratio of 1:1 with infected CD4 T cells as in a. Open symbols on the right of each panel indicate the percentage of Gag expression in the CD4 T cells if no CD8 T cells were added at all. Cells were harvested for analysis of percentage of Gag+ CD4 T cells (CD8− T cells) after 7 d of co-culture. Statistical analysis was performed using one-way ANOVA. (right) Representative Western blot analysis of Bim levels after knockdown, determined at 48 h after activation. (c) Purified CD8 T cells from viral controllers were nucleofected with either control RNA or TRAF1 siRNA and incubated with autologous monocytes that had been pulsed with control or HIV peptide and pretreated with replication defective adenovirus expressing 4-1BBL or CD80 at a multiplicity of infection of 200, as previously described (Bukczynski et al., 2004). 8 d later, the cells were harvested for FACS analysis. (top left) Representative Western blot analysis of TRAF1 levels at the end of the 8-d culture. (bottom) Representative FACS plots. (top right) Summary plot for three experiments with cells from two different donors, shown as the number of HIV-tetramer+ CD8 T cells recovered in the TRAF1 siRNA-transfected population over those transfected with control RNA after stimulation with HIV peptide-pulsed 4-1BBL or CD80-expressing monocytes.
Figure 3.

TRAF1 is required for HIV-specific CD8 T cell responses. (a) CD8 and CD4 T cells were separately purified from HIV+ or HIV− donors. CD4 T cells were infected with a primary isolate of HIV and at 48 h, co-cultured with their autologous CD8 T cells that had been transfected with either TRAF1-specific siRNA or a control scrambled RNA (ctrl). Irradiated autologous PBMCs were added as a source of APC. The frequency of Gag+ T cells was measured 5–7 d later by flow cytometry using CD3-, CD8-, and GAG-specific antibodies to assess the proportion of infected CD4 T cells. As CD4 is down-regulated on the infected cells, the absence of CD8 is used to determine the CD4 T cell population. Representative flow cytometry plots are shown in Fig. S2. (left and middle) representative suppression curves (based on three to five replicates at each effector-to-target ratio for each donor and representative of three viral controllers and a healthy uninfected control). Statistical significance was determined by linear regression of percentage of Gag+ T cells against log (effector: target ratio) using GraphPad (Prism) software. (right) Representative Western blot analysis of TRAF1 levels after knockdown, determined at 48 h after activation. (b). (left and middle) Viral suppression assay performed as in a. Bim-specific siRNA, TRAF1-specific siRNA, or both, or control scrambled RNA were used to transfect CD8 T cells from two viral controllers. CD8 T cells were plated at a ratio of 1:1 with infected CD4 T cells as in a. Open symbols on the right of each panel indicate the percentage of Gag expression in the CD4 T cells if no CD8 T cells were added at all. Cells were harvested for analysis of percentage of Gag+ CD4 T cells (CD8− T cells) after 7 d of co-culture. Statistical analysis was performed using one-way ANOVA. (right) Representative Western blot analysis of Bim levels after knockdown, determined at 48 h after activation. (c) Purified CD8 T cells from viral controllers were nucleofected with either control RNA or TRAF1 siRNA and incubated with autologous monocytes that had been pulsed with control or HIV peptide and pretreated with replication defective adenovirus expressing 4-1BBL or CD80 at a multiplicity of infection of 200, as previously described (Bukczynski et al., 2004). 8 d later, the cells were harvested for FACS analysis. (top left) Representative Western blot analysis of TRAF1 levels at the end of the 8-d culture. (bottom) Representative FACS plots. (top right) Summary plot for three experiments with cells from two different donors, shown as the number of HIV-tetramer+ CD8 T cells recovered in the TRAF1 siRNA-transfected population over those transfected with control RNA after stimulation with HIV peptide-pulsed 4-1BBL or CD80-expressing monocytes.

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